Structural Basis of PPARγ-Mediated Transcriptional Repression by the Covalent Inverse Agonist FX-909

Abstract

Hyperactivation of peroxisome proliferator-activated receptor γ-mediated transcription promotes tumor growth in urothelial (bladder) cancer, which can be inhibited by compounds that repress PPARγ activity. FX-909 is a covalent PPARγ inverse agonist in phase 1 clinical trials for advanced solid malignancies, including muscle-invasive bladder cancer. Here, we compared the mechanism of action of FX-909 to other covalent inverse agonists including T0070907, reported more than 20 years ago and misclassified as an antagonist, and two reported improved covalent inverse agonist analogs, SR33068 and BAY-4931. Functional profiling and NMR studies reveal that FX-909 displays improved corepressor-selective inverse agonism and better stabilizes a transcriptionally repressive PPARγ LBD conformation compared to T0070907. The crystal structure of PPARγ LBD cobound to FX-909 and the NCoR1 corepressor peptide reveals a repressive conformation shared by other covalent inverse agonists. These findings build on recent studies highlighting the pharmacological significance and clinical relevance of transcriptionally repressive PPARγ inverse agonists.

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Pharmacological PPARγ ligand set used in this study. (A) Chemical structures and pharmacological properties of the ligands. The halogen exchange reaction covalent leaving groups present in all compounds except rosiglitazone are shown in gray. (B) Transcriptional reporter assay performed in HEK293T cells transfected with a full-length PPARγ expression plasmid and a 3xPPRE-luciferase reporter plasmid. Data were normalized to cells treated with DMSO control, represented as mean ± s.d. (n = 4), and were fit to a three-parameter sigmoidal dose–response equation to obtain EC₅₀/IC₅₀ values.

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Biochemical coregulator profiling data. Time-resolved fluorescence resonance energy transfer (TR-FRET) coregulator interaction assay performed with (A) the NCoR1 corepressor peptide or (B) the TRAP220/MED1 coactivator peptide in the presence of the indicated ligands. Data represent mean ± s.e.m. (n = 3) and were fit to a four-parameter sigmoidal dose–response equation to obtain EC₅₀/IC₅₀ values. Fluorescence polarization (FP) binding assay performed with (C) NCoR1 corepressor peptide or (D) TRAP220/MED1 coactivator peptide in the presence of the indicated ligands. Data represent mean ± s.e.m. (n = 3) and were fit to a quadratic binding equation that assumes binding occurs in a titration binding regime to obtain K _d values. (E) Pairwise correlation plots between PPARγ-mediated transcription (y-axis) and biochemical coregulator profiling data (x-axis). Spearman correlation coefficients (r _p) and associated two-tailed p values are listed.

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Crystal structures of PPARγ LBD in transcriptionally repressive and active conformations. The repressive conformation crystal structures are bound to the NCoR1 corepressor peptide (purple) and covalent ligands including FX-909 (PDB 9O9N), T0070907 (PDB 6ONI), BAY-4931 (PDB 8AQN), SR33068 (PDB 8FKD), and GW9662 (PDB 8FHE). The active conformation crystal structure is bound to the TRAP220/MED1 coactivator peptide (dark blue) and rosiglitazone (PDB 6ONJ). The difference between the repressive and active states is most notable in the conformation of helix 3 (orange) and helix 12 (cyan). Ligands are colored according to their annotated names displayed below the structures.

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Ligand interactions in the crystal structures. Zoomed views are shown for PPARγ LBD bound to (A) FX-909 (PDB entry 9O9N), (B) T0070907 (PDB entry 6ONI), (C) BAY-4931 (PDB entry 8AQN), (D) SR33068 (PDB entry 8FKD), (E) GW9662 (PDB entry 8FHE), and (F) rosiglitazone (PDB entry 6ONJ). Secondary structural elements are annotated in gray (e.g., h3, h5, h7, h12). PPARγ LBD residues are annotated in black. Distances are annotated in yellow. Ligands are colored according to the figure panel titles.

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Peptide interactions in the crystal structures. Zoomed-in views are shown for PPARγ LBD bound to (A) FX-909 (PDB entry 9O9N), (B) T0070907 (PDB entry 6ONI), (C) BAY-4931 (PDB entry 8AQN), (D) SR33068 (PDB entry 8FKD), (E) GW9662 (PDB entry 8FHE), and (F) rosiglitazone (PDB entry 6ONJ). Secondary structural elements are annotated in gray (e.g., h3, h5, h7, h12). PPARγ LBD residues are annotated in black. Distances are annotated in yellow. Ligands and NCoR1/MED1 peptide residues are colored according to the figure panel titles.

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Helix 12 interactions in the repressive conformation crystal structures. Zoomed-in views are shown for the PPARγ LBD bound to (A) FX-909 (PDB 9O9N), (B) T0070907 (PDB 6ONI), (C) BAY-4931 (PDB 8AQN), (D) SR33068 (PDB 8FKD), and (E) GW9662 (PDB 8FHE). Secondary structural elements are annotated in gray (h3, β2) except for h12 (colored according to panel titles). PPARγ LBD residues are annotated in black. Distances are annotated in yellow. Ligands and helix 12 (h12) are colored according to the figure panel titles.

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NMR shows that FX-909 shifts the PPARγ LBD conformational ensemble toward a repressive conformation. (A) Structural location of two residues (D380 and G399) focused on in the NMR analysis. (B) Overlays of two-dimensional (2D) [¹H,¹⁵N]-TROSY-HSQC spectra of ¹⁵N-labeled PPARγ LBD zoomed into the backbone amides of the indicated residues (in A) in the absence or presence of the indicated ligands. In all panels, the green and pink gradient lines with arrows denote the colinear (G399) and noncolinear/vector-based (D380) shifting between ground state (white) and active (green) or repressive (pink) states caused by ligand binding.